RNA interference (RNAi) is an evolutionarily conserved mechanism whereby microRNAs and other double-stranded RNA molecules effect sequence-specific gene regulation. RNAi has been heavily used as a tool to manipulate gene expression in vitro as well as in vivo. Its potential as a therapeutic approach is also widely studied (Elbashir et al. 2001; Xia et al. 2002; Dorsett and Tuschl 2004; Xia et al. 2004; Harper et al. 2005; Amarzguioui et al. 2006; Behlke 2006; Bernards et al. 2006; Chang et al. 2006; Fewell and Schmitt 2006; Vlassov et al. 2006). Multiple RNA structures can be used to perform RNAi, including siRNAs, Dicer-substrate RNAs, long dsRNAs, small hairpin RNAs (shRNAs) either in synthetic or expressed form. Among synthetic RNAi triggers, we and others have found a special class of shRNA, short shRNAs (sshRNA) that have identical, or in some cases, slightly better efficacy than siRNAs that target the same sequences (Li et al. 2007; Vlassov et al. 2007; and U.S. Provisional Patent Application Ser. No. 61/105,606, filed Oct. 15, 2008, all of which are incorporated herein by reference in their entirety). As described therein, sshRNAs have a stem length of 19 bp or less (where the stem is formed from sense and antisense sequences for the target RNA of interest), a connection of 0 to 9 nt between the antisense and the sense sequence, and optionally a 1 to 2-base 3′-overhang. The connection (also called a loop) is sometimes preferred at the 3′ end of the antisense sequence (L sshRNAs) as opposed to being at the 3′ end of the sense sequence (R sshRNAs) for better RNAi activity (McManus et al. 2002; Harborth et al. 2003).
To promote in vivo applications of synthetic sshRNAs, several factors should be considered, including 1) the nuclease stability of RNA duplexes in biological fluids such as serum; 2) the delivery and cellular uptake of the RNA duplex with sufficient cell specificity and efficiency; 3) minimal undesired innate immune responses; 4) minimal off-target effects. For instance, various results have demonstrated the ability of synthetic siRNAs and shRNAs to activate mammalian immune responses (Kariko et al. 2004; Kim et al. 2004; Hornung et al. 2005; Judge et al. 2005; Sioud 2005; Marques et al. 2006; Schlee et al. 2006; Judge and MacLachlan 2008; Robbins et al. 2008, incorporated herein by reference in their entirety). Toll-like receptors (TLR3, TLR7, and TLR8), protein kinase R (PKR), the cytosolic RNA helicase retinoic acid-inducible gene (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) are involved in the synthetic RNAi molecule-mediated recognition and activation of the innate immune response (Judge and MacLachlan 2008) (incorporated herein by reference in its entirety). Several features of RNA, including length, sequence, and structure could account for the recognition by these receptors (Hornung et al. 2005; Judge et al. 2005; Forsbach et al. 2008), (incorporated herein by reference in their entirety). Although some level of inflammatory cytokine expression may be beneficial to antiviral or even anti-tumor therapeutics (Poeck et al. 2008) (incorporated herein by reference in its entirety), the toxicities associated with excessive cytokine release and associated inflammatory syndromes are an undesirable side-effect.
Small synthetic RNAs (such as siRNA and shRNA) that exploit the naturally existing RNA interference mechanism normally used by endogenous microRNAs are potent agents for controlling gene expression in human cells. To translate this potency into therapeutics, it is necessary to optimize the efficacy of the RNA-based drugs. Besides selecting effective RNA sequences, the optimization includes chemical modifications to improve their in vivo nuclease stability, cellular delivery, biodistribution, pharmacokinetics, potency, and specificity while reducing off-target effects and immune response. Various chemical modifications, most of which were originally developed for ribozymes and RNA aptamers, have been proposed in issued and pending patent applications for siRNAs (e.g. in U.S. Pat. No. 7,595,387; WO2004090105; US20060247428; US20070167384; US20070167393; US20090209626) as well as for synthetic (vs. unmodified vector-expressed) shRNAs (WO03070750; WO2004015075; US20040209831; US20060223777; US20070004665; U.S. Pat. No. 7,595,387). The modified siRNA and shRNA molecules include various modifications of sugar, internucleotide phospodiester bonds, purine and pyrimidine residues, as well as non-nucleotide links, bridges, loops and conjugates (Manoharan 2004; Corey 2007; Behlke, 2008; Watts et al. 2008; Shukla et al. 2010).
The future success of RNA-based drugs relies on the identification of appropriate chemical modifications placed at appropriate positions in these RNAs. Because of a lack of a clear mechanistic understanding of the effect of different modifications on messenger RNA (mRNA) silencing mediated by the RNA-induced silencing complex (RISC) (Skulka et al. 2010), there are not general rules for optimization of RNA-drugs. The effect of particular modifications strongly depends on the sequence and size of RNA drugs. For example, double-stranded RNA molecules acting through different pathways (e.g. acting either as Dicer or non-Dicer substrates) require different modification strategies (see, e.g., Pavco et al. WO2009102427A2). Similarly, short shRNAs (sshRNAs) cannot be processed by Dicer in contrast to ordinary shRNAs, and, therefore, must be processed by a Dicer-independent mechanism (Cifuentes et al. 2010). The difference between the mechanisms of nuclease-assisted processing of ordinary shRNAs and the sshRNAs requires different modification patterns, which do not interfere with the latter mechanism. Because of this difference in mechanism, the specific patterns for modifications of sshRNAs are not predictable based on the prior art.